Implant comprising oxidized cellulose and method for preparing such an implant

ABSTRACT

The present invention relates to an implant ( 10 ) comprising: -a substrate ( 1 ) the surface of which comprising oxidized cellulose, said oxidized cellulose having a degree of oxidation ranging from 0.0 to 1, and -a multilayer coating covering said substrate, said multilayer coating comprising at least a first layer ( 2 ) adjacent said substrate, said first layer being formed of chitosan, and a second layer ( 3 ) adjacent said first layer, said second layer being formed of oxidized cellulose having a degree of oxidation ranging from 0.5 to 1. The invention further relates to a method for preparing such an implant.

The present invention relates to implants comprising a substrate thesurface of which comprises oxidized cellulose and a coating applied onsaid substrate.

These implants may be used, for example, as haemostatic patches,adhesion prevention barriers, or as wall reinforcements in the repair,reinforcement or replacement of soft tissues.

Oxidized cellulose is of high interest in the field of medical implants.Oxidized cellulose is a highly biocompatible material, showingproperties such as high moisture, handling ability which can help therepair, reinforcement or replacement of soft tissues.

Oxidized cellulose has bactericidal effects towards a wide spectrum ofaerobic and anaerobic pathogens and therefore shows a therapeuticpotential for use in medical implants in general.

In addition, thanks to its good haemostatic effects, biosolubility andbiodegradability, antioxidant and wound-healing properties, oxidizedcellulose represents a suitable means for the therapy of bleedingconditions in various fields of medicine. As such, oxidized cellulosemay advantageously constitute the absorbent substrate of a haemostaticpatch.

Anyway, it has been observed that non bioresorbable oxidized cellulosecould trigger an undesirable immune response from the body onceimplanted. On the contrary, for some applications, bioresorbableoxidized cellulose degrades too rapidly in physiological conditions: themechanical properties of the implant disappear before the implant hadtime to perform its intended function, thereby jeopardizing the successof the surgical operation.

For the purpose of the present application, the term “bioresorbable” isintended to mean the characteristic according to which a material isdegraded by the biological tissues and the surrounding fluids, in vivoafter a given period of time, that may vary, for example, from a fewhours to several months, depending on the chemical nature of thematerial.

Haemostatic patches are implantable medical devices for stopping theflow of biological fluids such as blood during surgery. Permanentimplants are not always necessary for use as haemostatic patches. Forexample, bioresorbable or partly-bioresorbable haemostatic patches maybe made to at least partly disappear in vivo after implantation, after afew weeks for example, when their function of stopping the effusion ofbiological fluids is no longer necessary.

Permanent implants are neither always essential for the repair,reinforcement or replacement of soft tissues. For example, in the caseof treatment of certain defects such as for the treatment of hernias orreconstruction of a visceral wall, one may seek to limit the amount offoreign bodies which remain permanently in a human body and promotetissue reconstruction.

Thus, it would be desirable to provide an implant provoking no immuneresponse from the body once implanted, this implant having neverthelessa substrate comprising oxidized cellulose, where this oxidized cellulosewould show a degradation time sufficiently delayed so that the implantwould be capable of performing its intended function.

The Applicant has found that by applying a particular coating on asubstrate the surface of which comprises a specific oxidized cellulose,it was possible to obtain an implant comprising oxidized cellulosecapable of having a degradation time in vivo sufficient for obtainingthe intended results expected from the presence of oxidized cellulosewhile avoiding problematic immune response from the body.

A first aspect of the invention is an implant comprising:

-   -   a substrate the surface of which comprising at least oxidized        cellulose, said oxidized cellulose having a degree of oxidation        ranging from 0.5 to 1, and    -   a multilayer coating covering said substrate, said multilayer        coating comprising at least a first layer adjacent the surface        of said substrate, said first layer being formed of chitosan,        and a second layer adjacent said first layer, said second layer        being formed of oxidized cellulose having a degree of oxidation        ranging from 0.5 to 1.

The implant of the invention is at least partly bioresorbable. In theimplant of the invention, the oxidized cellulose present at least at thesurface of the substrate is bioresorbable and shows a degradation timein physiological conditions compatible with the function the implant isintended to perform. The implant of the invention therefore allowsproviding at least partly bioresorbable implants comprising oxidizedcellulose having both a significant degradation time in vivo andfurthermore showing the properties of oxidized cellulose in general,such as biocompatibility, antioxidant and haemostatic properties,bactericidal properties, etc . . . In particular, the implants of theinvention show good haemostatic properties and good mechanicalproperties.

Another aspect of the invention is a method for preparing an implant asdescribed herein, comprising the following steps:

-   -   i) providing a substrate the surface of which comprising at        least oxidized cellulose, said oxidized cellulose having a        degree of oxidation ranging from 0.5 to 1,    -   ii) providing a solution of chitosan,    -   iii) providing a solution of oxidized cellulose having a degree        of oxidation ranging from 0.5 to 1,    -   iv) covering said substrate with a part of the solution of        chitosan of ii) for forming a first layer adjacent the surface        of said substrate,    -   v) covering the one layer coated substrate obtained in step iv)        with a part of the solution of oxidized cellulose of iii) for        forming a second layer adjacent said first layer,    -   vi) optionally repeating steps iv) and v) as many times as        desired.

The advantages of the invention will be better understood in view of thedetailed description that follows and with the help of the accompanyingdrawings in which:

FIGS. 1A-1E are cross section views showing the preparation of anembodiment of an implant according to the invention, with a first layerformed of chitosan and a second layer formed of oxidized cellulose,

FIGS. 2A-2B are cross section views showing the preparation of anotherembodiment of an implant of the invention, provided with a third layer,formed of chitosan.

The implant of the present invention comprises a substrate the surfaceof which comprises at least oxidized cellulose, said oxidized cellulosehaving a degree of oxidation ranging from 0.5 to 1.

By “degree of oxidation” of the oxidized cellulose, is meant in thepresent application, the ratio of oxidized C₆ primary alcohol groups tooxidizable C₆ primary alcohol groups. In particular, in the oxidizedcellulose suitable for the present invention, from 50% to 100% of theoxidizable C₆ primary alcohol groups are oxidized. In embodiments wherethe degree of oxidation is 1, and therefore 100% of the oxidizable C₆primary alcohol groups are oxidized, the oxidized cellulose ispolyglucuronic acid.

Oxidized cellulose is a known material. Non oxidized cellulose comprisesa plurality of anhydroglucose units, each anyhdroglucose unit comprisingthree hydroxyl groups. From these three hydroxyl groups, two of them aresecondary alcohols (positioned on carbons C₂ and C₃ of theanhydroglucose unit) and one is a primary alcohol (positioned on carbonC₆ of the anydroglucose unit). In the present application, theoxidizable C₆ primary alcohol groups are the C₆ primary alcohol groupsinitially present in the cellulose before oxidation. Oxidized cellulosesuitable for the present invention is oxidized cellulose, where the C₆primary alcohol is partially or fully oxidized to carboxylic acid, forexample to give polyglucuronic acid, and mixtures thereof. Oxidizedcellulose suitable for the present invention is oxidized cellulose,where the C₆ primary alcohol groups are partially or fully oxidized tocarboxylic acid groups. In particular, in the oxidized cellulosesuitable for the invention, the C₂ and C₃ secondary alcohol groups arenot oxidized. Because the oxidized cellulose suitable for the inventioncomprises carboxylic acid groups, said oxidized cellulose is negativelycharged. The cellulose used for oxidation can be obtained from type Icellulose or can be regenerated.

Several types of regenerated cellulose have been developed industrially.We may mention for example the “viscose” process, which is based on thesolubility of cellulose xanthate in a dilute solution of sodiumhydroxide. We may also mention the so-called “cupro-ammonium process”employed for example by the company Bemberg in Italy or the companyAsahi Chemical Industries in Japan, which consists of dissolvingcellulose in an ammoniacal solution of copper. Another method ofpreparing regenerated cellulose suitable for the present invention isthe method of organic-phase dissolution of cellulose byN-methylmorpholine oxide (N.M.M.O.), called the “Lyocell® process”,employed for example by the company Lenzing in Austria.

The oxidized cellulose of the substrate of the implant of the inventionshows a degree of oxidation ranging from 0.5 to 1, wherein the degree ofoxidation is the ratio of oxidized C₆ primary alcohol groups tooxidizable C₆ primary alcohol groups. An oxidized cellulose with such adegree of oxidation is bioresorbable. The oxidized cellulose suitablefor the present invention may be prepared by oxidation of cellulose withNO₂. Oxidation of cellulose with NO₂ is a selective reaction allowingthe oxidation to occur on the C₆ primary alcohol group only, so that thehydroxyl group of carbon C₆ is oxidized into a carboxylic acid group.The preparation of such an oxidized cellulose is described inWO2006/018552. The oxidized cellulose of the present invention, inparticular prepared as described in WO2006/018552, is a cellulose wherepart or all of the C₆ primary alcohol groups are oxidized to carboxylicacid groups, said oxidized cellulose being therefore negatively charged.

Oxidized cellulose of degree of oxidation ranging from 0.5 to 1 asdefined above may also be commercially available from company Ethiconunder the tradename of “Surgicel®” or “Interceed®”, or from companyCovidien under the tradename of “Veriset®”.

In embodiments, the oxidized cellulose suitable for the presentinvention present at least at the surface of the substrate of theimplant of the invention shows a degree of oxidation of about 0.6 asdefined above. Such an oxidized cellulose is amorphic. In embodiments,for example, when it is desired to maximize the haemostatic efficiencyof the oxidized cellulose, the oxidized cellulose shows a degree ofoxidation, as defined above, ranging from 0.6 to 1.

Oxidized cellulose suitable for the present invention, where the C₆primary alcohol groups are partially or fully oxidized to carboxylicacid groups, is a compound which is naturally negatively charged. Thesurface of the substrate of the implant of the invention is thereforenegatively charged. In this view, the substrate of the implant of theinvention may optionally further comprise other biocompatiblebioresorbable or non bioresorbable materials in addition to oxidizedcellulose of degree of oxidation, as defined above, ranging from 0.5 to1, as long as its surface remains negatively charged. In particular, thesubstrate of the implant of the invention may further comprise a neutralmaterial or a negatively charged material.

Suitable neutral non bioresorbable materials that may be included in thesubstrate of the implant of the invention may be selected frompolypropylene, polyethylene terephthalate and combinations thereof.Suitable neutral bioresorbable materials that may be included in thesubstrate of the implant of the invention may be selected frompolylactic acid, polyglycolic acid and combinations thereof. Suitablebioresorbable negatively charged materials that may be included in thesubstrate of the implant of the invention may be selected frompolysaccharides such as alginate, hyaluronan and combinations thereof.

In embodiments, the implant of the invention is bioresorbable. Forexample, the substrate consists in bioresorbable materials only. Apartfrom the oxidized cellulose of degree of oxidation, as defined above,ranging from 0.5 to 1, these bioresorbable materials may be selectedfrom polylactic acid, polyglycolic acid, polysaccharides such asalginate, hyaluronan, and combinations thereof. In embodiments, thesubstrate of the implant of the invention consists in said oxidizedcellulose having a degree of oxidation ranging from 0.5 to 1, as definedabove, namely wherein the degree of oxidation is the ratio of oxidizedC₆ primary alcohol groups to oxidizable C₆ primary alcohol groups. Noother material is present in the substrate or at the surface of thesubstrate. In such embodiments, the implant of the invention isbioresorbable. The implant of the invention therefore shows adegradation time in vivo greater than the degradation time that would bethat of the substrate taken on its own. The implant of the inventiontherefore allows providing fully bioresorbable implants comprisingoxidized cellulose having both a significant degradation time in vivoand furthermore showing the properties of oxidized cellulose in general,such as biocompatibility, antioxidant and haemostatic properties,bactericidal properties, etc . . .

The substrate of the implant of the present invention may show any form,shape or structure suitable for implantation inside a human or animalbody, in adequation with the function the implant is intended toperform.

For example, the substrate may be under the form of porous or solidbody. In a view of being used for forming either a haemostatic patch ora reinforcement wall, the substrate may preferably be under the form ofa porous layer. In embodiments, the substrate is under the form of aporous layer.

In the present application, “porous layer” means a layer having pores,or voids, cells, holes, orifices, distributed regularly or irregularly,not only on the surface but also within the thickness of said layer, andmore or less interconnected. Said porous layer is particularly effectivefor absorbing biological fluids such as blood and may be useful forforming a haemostatic patch. Such a porous layer is also effective forpromoting cell colonization and may be useful for forming areinforcement wall in hernia repair.

Such a porous layer may for example be under the form of a foam or asponge, for example obtained by freeze-drying of a solution of oxidizedcellulose, or under the form of an openwork textile.

According to the present application, “textile” means any arrangement orassemblage of biocompatible threads, fibres, filaments and/ormultifilaments, for example obtained by knitting, weaving, braiding, oralternatively non-woven. The arrangement of threads of the textileaccording to the invention defines at least two opposite faces, a firstface and a second face.

In the present application, “openwork textile” means any textile whosearrangement of threads of which it is constituted determines openings,cells or voids in the thickness of the textile and on the faces of thetextile, and these openings, cells or voids can constitute channels withopenings on either side of the textile. This openwork textile gives goodabsorption of biological fluids. Such an openwork textile also favorscell colonization.

In embodiments, the substrate is under the form of an openwork textile.The openwork textile may comprise yarns of oxidized cellulose having adegree of oxidation, as defined above, ranging from 0.5 to 1 andoptionally additional yarns of biocompatible bioresorbable or nonbioresorbable materials. In embodiments, the substrate consists in anopenwork textile of oxidized cellulose having a degree of oxidationranging from 0.5 to 1 as defined above, namely wherein the degree ofoxidation is the ratio of oxidized C₆ primary alcohol groups tooxidizable C₆ primary alcohol groups.

The openwork textile may be in the form of a two-dimensional orthree-dimensional knitted fabric.

“Two-dimensional knitted fabric” means, in the sense of the presentapplication, a knitted fabric having two opposite faces joined togetherby stitches but lacking cross-members giving it a certain thickness: aknitted fabric of this kind can be obtained for example by knittingthreads on a warp knitting machine or Raschel machine using two guidebars. Examples of knitting of two-dimensional knitted fabrics suitablefor the present invention are given in document WO2009/071998.

“Three-dimensional knitted fabric” means, according to the presentapplication, a knitted fabric having two opposite faces joined togetherby a cross-member giving the knitted fabric a significant thickness,said cross-member itself being formed of additional connecting threadssupplementary to the threads forming the two faces of the knittedfabric. This knitted fabric can be obtained for example on a warpknitting machine or double-bed Raschel machine using several guide bars.Examples of knitting of three-dimensional knitted fabrics suitable forthe present invention are given in documents WO99/05990, WO2009/031035,WO2009/071998.

This kind of three-dimensional knitted fabric, with the presence of aspacer giving it a significant thickness, provides excellent absorptionof biological fluids such as blood and is particularly suitable formanufacture of a haemostatic patch. Such a three-dimensional knittedfabric may alternatively be suitable for the manufacture of areinforcement wall, as its porosity and significant thickness providesfor an adequate medium and space for cell colonization.

A knitted fabric, in particular three-dimensional, based on oxidizedcellulose, can be obtained by knitting firstly threads of unoxidizedregenerated cellulose, then submitting the knitted fabric thus obtainedto oxidation.

In fact, when spun through a perforated plate, viscose sets in an acidmedium and forms long continuous filaments of regenerated cellulose,which are dried and combined in multifilament threads. A regeneratedcellulose thread is obtained that has good mechanical strength.

Generally this regenerated cellulose thread is not bioresorbable.However, it has good mechanical strength allowing it to be used formanufacturing a knitted fabric. As an example, we may mention, asregenerated cellulose thread suitable for manufacturing a knitted fabricsuitable for the porous layer of the patch of the invention, the 90decitex multifilament thread marketed under the name “CUPRO® Cusio” bythe Italian company Bemberg.

The knitted fabric obtained is then oxidized in order to form a porouslayer based on oxidized cellulose suitable for forming the substrate ofthe implant according to the present invention. In particular, theknitted fabric may be oxidized with NO₂ as described in WO2006/018552.Such a knitted fabric may show a high porosity allowing tissueintegration and is therefore particularly useful for the repair,reinforcement or replacement of soft tissues. Such a knitted fabric alsoshows good tensile strength allowing it to be fixed to tissues by anyknown techniques, such as suturing, stitching, stapling, tacking, andcombinations thereof. In addition, as a textile, such a knitted fabricis easy to handle and may further be formed in a specific desired shape.

The implant of the invention further comprises a multilayer coatingcovering the substrate. The multilayer coating comprises at least afirst layer adjacent the surface of said substrate, this first layerbeing formed of chitosan, and a second layer adjacent the first layer,this second layer being formed of oxidized cellulose having a degree ofoxidation ranging from 0.5 to 1 as defined above, namely wherein thedegree of oxidation is the ratio of oxidized C₆ primary alcohol groupsto oxidizable C₆ primary alcohol groups.

Chitosan is a biocompatible biopolymer obtained by deacetylation ofchitin. Chitin is extracted from exoskeletons of arthropods such as thelobster, crab, prawn, the endoskeleton of cephalopods such as the squid,or from fungi. Extraction of chitin involves steps of hydrolysis of theproteins and lipids, depigmentation and demineralization. Hydrolysis ofthe proteins and lipids is usually carried out in the presence of sodiumhydroxide, and demineralization requires the use of hydrochloric acid.

Once the chitin has been extracted, chitosan is obtained by adeacetylation step, which consists of hydrolysis of the acetamidegroups. This reaction is generally carried out at high temperature in analkaline solution, for example a 48% solution of sodium hydroxide (NaOH)in water, at 90° C. Chitosan is a compound that is soluble in aqueoussolution and can have a degree of acetylation (DA) of up to 70%.

The following publications describe processes for deacetylation ofchitin to obtain chitosan: “Lamarque, G., C. Viton, and A. Domard, NewRoute of Deacetylation of α- and β-Chitins by means of Freeze-PumpOut-Thaw Cycles. Biomacromolecules, 2005. 6(3): p. 1380-1388.”,“Lamarque, G., C. Viton, and A. Domard, Comparative Study of the FirstHeterogeneous Deacetylation of α- and β-Chitins in a Multistep Process.Biomacromolecules, 2004. 5(3): p. 992-1001.”, “Lamarque, G., C. Viton,and A. Domard, Comparative Study of the Second and Third HeterogeneousDeacetylations of α- and β-Chitins in a Multistep Process.Biomacromolecules, 2004. 5(5): p. 1899-1907.”, “Tolaimate, A., et al.,Contribution to the preparation of chitins and chitosans with controlledphysicochemical properties. Polymer, 2003. 44(26): p. 7939-7952.”

Chitosan is a bioresorbable compound. The degree of acetylation ofchitosan can have an influence on the kinetics of degradation ofchitosan. Thus, depending on the kinetics of biodegradation desired forthe implant of the invention, the chitosan will have to have a degree ofacetylation ranging from 0 to 70%.

In embodiments, the degree of acetylation of the chitosan is 30%.

The oxidized cellulose used for forming the second layer of themultilayer coating of the substrate of the implant of the invention maybe defined in the same way as the oxidized cellulose defined above forthe substrate of the implant. In particular, the oxidized cellulose usedfor forming the second layer of the multilayer coating of the substrateof the implant of the invention is a cellulose where the C₆ primaryalcohol groups are partially or fully oxidized to carboxylic acid groupsand is a negatively charged compound. This oxidized cellulose may beprepared as described in WO2006/018552. This oxidized cellulose shows adegree of oxidation ranging from 0.5 to 1, wherein the degree ofoxidation is the ratio of oxidized C₆ primary alcohol groups tooxidizable C₆ primary alcohol groups, and is bioresorbable. Inembodiments, this oxidized cellulose shows a degree of oxidation asdefined above of 0.6. Such an oxidized cellulose is amorphic. Inembodiments, the oxidized cellulose shows a degree of oxidation asdefined above ranging from 0.6 to 1.

In embodiments, the multilayer coating further comprises a third layeradjacent the second layer, the third layer being formed of chitosan. Thechitosan used for forming the third layer of the multilayer coating ofthe substrate of the implant of the invention may show the same degreeof acetylation as the chitosan used for the first layer or on thecontrary another degree of acetylation. The choice of the degree ofacetylation of the chitosan for the first layer and of the chitosan ofthe third layer may depend on the desired degradation profile for theimplant once implanted. An implant with a three-layer coating, namely afirst layer of chitosan, a second layer of oxidized cellulose and athird layer of chitosan shows a prolonged degradation time in vivo withrespect to an implant with no coating or that would be coated with onelayer of chitosan only for example. It is therefore possible to providean implant comprising oxidized cellulose and showing the properties ofoxidized cellulose having in addition a delayed degradation time in vivoand therefore capable of performing its function of either a haemostaticpatch or a wall reinforcement during a significant time. In particular,it is possible to provide implants comprising oxidized cellulose, havinga delayed degradation time in vivo, and further showing good haemostaticproperties and good mechanical properties. In further embodiments, themultilayer coating further comprises a fourth layer adjacent the thirdlayer, said fourth layer being formed of oxidized cellulose having adegree of oxidation ranging from 0.5 to 1. In embodiments, themultilayer coating further comprises a fifth layer adjacent the fourthlayer, said fifth layer being formed of chitosan. Such embodiments ofthe implant of the invention allow ensuring a long time performance ofthe oxidized cellulose present at least at the surface of the substrate.

The method for preparing the implant of the invention comprises thefollowing steps:

-   -   i) providing a substrate the surface of which comprising at        least oxidized cellulose, said oxidized cellulose having a        degree of oxidation ranging from 0.5 to 1,    -   ii) providing a solution of chitosan,    -   iii) providing a solution of oxidized cellulose having a degree        of oxidation ranging from 0.5 to 1,    -   iv) covering said substrate with a part of the solution of        chitosan of ii) for forming a first layer adjacent the surface        of said substrate,    -   v) covering the one layer coated substrate obtained in step iv)        with a part of the solution of oxidized cellulose of iii) for        forming a second layer adjacent said first layer,    -   vi) optionally repeating steps iv) and v) as many times as        desired.

The multilayer coating of the implant of the invention is manufacturedby applying the first, second and optionally third, fourth and fifthlayers on the substrate by electrostatic layer-by-layer self assembly(“ELBL”). ELBL is a technique in which layers are assembled byalternating the adsorption of oppositely-charged polyelectrolytes. Theprocess is based on the reversal of the surface charge of the coatingafter the deposition of each layer.

As seen above, the surface of the substrate comprising oxidizedcellulose suitable for the present invention is negatively charged.Chitosan is a polycationic material and therefore a positively chargedcompound. A first layer of chitosan is applied on the surface of thesubstrate comprising oxidized cellulose as defined above. The coating ofthe substrate therefore becomes positively charged. A second layer, ofnegatively charged oxidized cellulose, as defined above, is then appliedon the first layer of chitosan. The coating becomes negatively charged.A third layer, of positively charged chitosan is then applied on thesecond layer. The coating becomes positively charged again.

The physical basis of association is electrostatics. The application ofalternately charged layers may be repeated as many times as needed.

For the manufacture of the multilayer coating as describe above,solutions of chitosan and solutions of oxidized cellulose as definedabove are prepared, and the substrate may be successively covered byparts of these solutions.

The chitosan solution is generally prepared starting from a solution ofchitosan in acidic water. The concentration of chitosan in said solutionmay range for example from 0.01% to 1%, by weight, relative to the totalweight of the solution. The concentration of chitosan in the solutionmay depend on the molecular weight of the chitosan used. For example,for chitosan of high molecular weight, the concentration may be chosenin the low part of the range given above, so that the molecules ofchitosan have enough space not to become entangled, and therebyefficiently leading to the formation of a complex between the variouslayers of the coating of the implant of the invention. Sodium chloride(NaCl) may be added to the solution. The pH of the solution usuallyranges from 1.5 to 7.

The oxidized cellulose solution is generally prepared by dissolvingoxidized cellulose as defined above in water. Caustic soda may be addedto the solution. The concentration of oxidized cellulose in saidsolution may range for example from 0.01% to 1%, by weight, relative tothe total weight of the solution. Acid, such as chlorhydric acid, may beadded to the solution in order to adjust the pH from 3 to 7. Theoxidized cellulose concentration is determined in function of theintended degradation profile of the layer which will be formed. Sodiumchloride (NaCl) may be added to the solution.

In embodiments, step iv) comprises dipping the substrate in the solutionof chitosan of ii), and step v) comprises dipping the one layer coatedsubstrate obtained in step iv) in the solution of oxidized cellulose ofiii).

The substrate is for example dipped in the chitosan solution for adetermined time and then retrieved from the solution. The time duringwhich the substrate is dipped into the solution should be sufficient forallowing the amount of solution necessary for further forming a coatingto contact and attach to the negatively charged surface of thesubstrate. For example, this time may range from 5 to 60 minutes. Inembodiments, this time is about 15 minutes. The substrate coated withthis first layer is then washed, for example in water. This first layermay show a thickness ranging from 0.5 to 10 nm.

The substrate is then for example dipped in the oxidized cellulosesolution for a determined time and then retrieved from the solution.Like for the first layer, the time during which the one layer coatedsubstrate is dipped into the solution should be sufficient for allowingthe amount of solution necessary for further forming a coating tocontact and attach to the positively charged surface of the first layerof the now coated substrate. For example, this time may range from 1 to60 minutes. In embodiments, this time is about 15 minutes. The substratecoated with the first and second layers is then washed, for example inwater. The second layer may show a thickness ranging from 0.5 to 10 nm.

These operations may be repeated as many times as necessary, alternatingthe dipping of the two/three/four/. . . -layer coated substrate in thechitosan solution and in the oxidized cellulose solution.

In other embodiments, each layer may be applied on the substrate and/orthe previous layer by a brush previously dipped in the solution of thecomponent, chitosan or oxidized cellulose as defined above, intended tobe applied. For example, step iv) comprises applying on said substratesaid solution of chitosan of ii) by means of a brush previously dippedin said solution of chitosan, and step v) comprises applying on said onelayer coated substrate obtained in step iv) the solution of oxidizedcellulose of iii) by means of a brush previously dipped in said solutionof oxidized cellulose.

Alternatively, in other embodiments, each solution of a component,chitosan or oxidized cellulose as defined above, may be sprayed onto thesubstrate and/or previous layer. For example, step iv) comprisesspraying on said substrate said solution of chitosan of ii), and step v)comprises spraying on said one layer coated substrate obtained in stepiv) the solution of oxidized cellulose of iii).

The implants of the invention may be used as bioresorbable orpartly-bioresorbable wall reinforcements, for example, in the repair,reinforcement or replacement of soft tissues when a permanent implant isnot necessary, e.g. treatment of hernias, reconstruction of a wall, suchas a visceral wall. They may also be used as haemostatic patches inorder to stop blood bleeding. The implants of the invention show goodhaemostatic properties and good mechanical properties.

The implants of the invention may also be used in vitro as a tissueengineering product or support for culturing live cells.

The implants of the invention may also be used as a barrier against thepost-surgical adhesions.

The examples that follow illustrate the invention.

EXAMPLES Example 1 Preparation of a Chitosan Solution: Solution A

0.111 g of chitosan (degree of acetylation 30%, molecular weight of 500000 g/mol) is dissolved in 100 g of water with a stoechiometric amountof acetic acid (CH₃COOH) in order to obtain a solution of chitosan at0.1% and pH 5.

Preparation of an Oxidized Cellulose Solution: Solution B

288.7 mg of oxidized cellulose (degree of oxidation 0.6 as definedabove) prepared according to WO2006/018552, is dissolved in 100 g ofwater together with 1.213 g of soda (NaOH). The pH of the solution is11.8. Chlorhydric acid (HCl) at 0.2 M is added in order to obtain asolution of oxidized cellulose at 1 g/l and pH 5.

Sodium chloride (NaCl) is added to each of the solution above in orderto obtain a final concentration of the component (chitosan or oxidizedcellulose) in the solution of 0.25 M.

A sample textile of oxidized cellulose (degree of oxidation 0.6 asdefined above), prepared according to WO2006/018552, of 3 cm×3 cm suchas the textile 1 shown on FIG. 1A is provided as a substrate forpreparing an implant. The textile 1 is preferably an openwork textile.The surface of the textile 1 is negatively charged. The substrateconsists in said textile and is therefore bioresorbable.

The textile 1 is dipped during 15 minutes in Solution A as shown on FIG.1B. The textile 1 is then removed from Solution A and washed during 1min in water. This washing step is repeated twice.

A textile 1 coated with a first layer 2 of chitosan is obtained, asshown on FIG. 1C.

The surface 2 a of the first layer 2 is positively charged.

The textile 1 coated with the layer 2 of chitosan is then dipped inSolution B during 15 minutes, as shown on FIG. 1D. The textile 1 is thenremoved from Solution B and washed during 1 min in water. This washingstep is repeated twice.

A textile 1 coated with a first layer 2 of chitosan and a second layer 3of oxidized cellulose is obtained, as shown on FIG. 1E. This coatedtextile forms an implant 10 according to the invention.

A first sample of an implant 10, herein after referred to as Implant A,is kept for tests described below in Example 2.

Another sample of an implant 10 is then dipped again in Solution Aduring 15 minutes, as shown on FIG. 2A. The sample textile is thenremoved from Solution A and washed during 1 min in water. This washingstep is repeated twice.

A textile 1 coated with a first layer 2 of chitosan, a second layer 3 ofoxidized cellulose and a third layer 4 of chitosan is obtained, as shownon FIG. 2B. This coated textile forms another implant according to theinvention, namely an implant 11. A sample of the implant 11, hereinafter referred to as Implant B, is kept for tests described below inExample 2.

Implant A and Implant B may be used as haemostatic patches or asadhesion prevention barriers. Implant A and B are bioresorbable.

In other embodiments not described, each layer may be applied on thetextile and/or the previous layer by a brush previously dipped in thesolution of the component, chitosan or oxidized cellulose, intended tobe applied. Alternatively, each solution of a component, chitosan oroxidized cellulose, may be sprayed onto the textile and/or previouslayer.

Example 2

In addition to Implant A and Implant B of Example 1, a non coated sampletextile 1 of oxidized cellulose, such as the one of Example 1, isprovided (herein after referred to as Textile A).

Implants A and B (invention) and Textile A (comparative) are immersed ina phosphate buffered saline (close to physiological fluid) solution.

After three days of immersion, Textile A has completely disappeared. Onthe contrary Implants A and B are still present in the phosphatebuffered saline solution.

After seven days of immersion, Implants A and B are still present in thephosphate buffered saline solution.

The multilayer coated implants (Implants A and B) of the invention allowproviding a bioresorbable or partly bioresorbable implant comprisingoxidized cellulose, and thus showing the properties of oxidizedcellulose, with a prolonged lifetime in a phosphate buffered salinesolution, and thus in physiological fluids such as those present in ahuman body when the implant is implanted.

1-14. (canceled)
 15. An implant comprising: a substrate comprising asurface comprising at least oxidized cellulose, said oxidized cellulosehaving a degree of oxidation ranging from 0.5 to 1, and a multilayercoating covering said substrate, said multilayer coating comprising atleast a first layer adjacent said surface of said substrate, said firstlayer being formed of chitosan, and a second layer adjacent said firstlayer, said second layer being formed of oxidized cellulose having adegree of oxidation ranging from 0.5 to
 1. 16. The implant according toclaim 15, wherein said substrate is a porous layer.
 17. The implantaccording to claim 15, wherein said substrate is an openwork textile.18. The implant according to claim 15, wherein said chitosan has adegree of acetylation of 30%.
 19. The implant according to claim 15,wherein said multilayer coating further comprises a third layer adjacentsaid second layer, said third layer being formed of chitosan.
 20. Theimplant according to claim 19, wherein said multilayer coating furthercomprises a fourth layer adjacent said third layer, said fourth layerbeing formed of oxidized cellulose having a degree of oxidation rangingfrom 0.5 to
 1. 21. The implant according to claim 20, wherein saidmultilayer coating further comprises a fifth layer adjacent said fourthlayer, said fifth layer being formed of chitosan.
 22. The implantaccording to claim 15, wherein said implant is bioresorbable.
 23. Theimplant according to claim 22, wherein said substrate consists of saidoxidized cellulose having a degree of oxidation ranging from 0.5 to 1.24. The implant according to claim 23, wherein said substrate consistsof an openwork textile of oxidized cellulose having a degree ofoxidation ranging from 0.5 to
 1. 25. A method for preparing an implantaccording comprising i) providing a substrate comprising a surfacecomprising at least oxidized cellulose, said oxidized cellulose having adegree of oxidation ranging from 0.5 to 1, ii) providing a solution ofchitosan, iii) providing a solution of oxidized cellulose having adegree of oxidation ranging from 0.5 to 1, iv) covering said substratewith a part of said solution of chitosan of ii) for forming a firstlayer adjacent said surface of said substrate, v) covering the one layercoated substrate obtained in iv) with a part of said solution ofoxidized cellulose of iii) for forming a second layer adjacent saidfirst layer, vi) optionally repeating iv) and v) as many times asdesired.
 26. The method according to claim 25, wherein iv) comprisesdipping said substrate in said solution of chitosan of ii), and v)comprises dipping said one layer coated substrate obtained in iv) insaid solution of oxidized cellulose of iii).
 27. The method according toclaim 25, wherein iv) comprises applying on said substrate said solutionof chitosan of ii) by means of a brush previously dipped in saidsolution of chitosan, and v) comprises applying on said one layer coatedsubstrate obtained in iv) said solution of oxidized cellulose of iii) bymeans of a brush previously dipped in said solution of oxidizedcellulose.
 28. The method according to claim 25, wherein iv) comprisesspraying on said substrate said solution of chitosan of ii), and v)comprises spraying on said one layer coated substrate obtained in iv)said solution of oxidized cellulose of iii).
 29. A method for preparingan implant according comprising i) covering a surface of a substratewith a solution of chitosan to form a first layer on the surface of thesubstrate, said surface of said substrate comprising at least oxidizedcellulose having a degree of oxidation ranging from 0.5 to 1, ii)covering said first layer on said surface of said substrate with asolution of oxidized cellulose to form a second layer adjacent saidfirst layer, iii) optionally repeating i) and ii) as many times asdesired.